Creatinine is the end metabolite when creatine becomes creatine phosphate and is used as the energy source for muscle contraction. This creatinine is filtered by the kidney glomeruli and then excreted into the urine without reabsorption. The determination of creatinine in body fluids is useful for diagnosing muscle diseases or various kidney diseases such as nephritis and renal insufficiency.
The first practical test for the determination of creatinine in urine, known as the Jaffe method, involves the formation of the red-yellowish brown colored creatinine picrate by the bonding of picric acid and creatinine in an alkaline solution. A more recent method for creatinine determination is reported by Benedict and Behre J. Biol. Chem., 113:515 (1936) which involves the reaction of 3,5-dinitrobenzoic acid with creatinine in an alkaline medium. The colorimetric determination of creatinine requires a high pH, on the order of from about 11.5 to 12.5, in order to deprotenate the creatinine enabling it to form the colored reaction product. Strongly basic substances such as alkali and alkaline earth metal hydroxides are typically used to maintain a suitably high pH in the reagent systems.
In jointly assigned U.S. Pat. No. 5,385,847 there is disclosed a unitary method for the determination of urinary protein and creatinine in a single urine sample. In this sort of assay the creatinine determination is used to minimize the problem,of high urine flow by using the protein/creatinine ratio to normalize the urine concentration. There is described in this disclosure a method for the unitary determination of urinary protein and creatinine in which a urine sample is introduced into a reaction vessel containing first and second reaction zones. The first reaction zone is incorporated with a dry immunoreagent specific for the urinary protein and the second reaction zone is incorporated with a dry basic reagent capable of raising the pH of a reaction fluid introduced into the reaction zones to a level suitable for creatinine determination. When the urine sample is introduced into the first reaction zone and brought into contact with the immunoreagent and a reaction fluid containing a polymeric agglutinator, it dissolves the immunoreagent causing an increase in the turbidity of the reaction fluid due to the interaction of the immunoreagent and the urinary protein which increase in turbidity can be used to determine the concentration of the urinary protein. Next, the reaction fluid, which contains a reagent for the determination of creatinine is brought into contact with the dry basic reagent in the second reaction zone to thereby dissolve the basic reagent and raise the pH of the reaction fluid to a level necessary for the colorimetric determination of creatinine. Alternatively, the creatinine determining reagent is pre-dried onto the surface of the second reaction zone along with the basic reagent. In order for this system of unitary analysis to operate at peak efficiency, it is essential that the dry creatinine reactive reagent be dried onto the surface of the second reaction zone in sufficient quantity to facilitate the creatinine determining reaction upon rehydration of this reagent.
The use of lithium salts in analytical procedures is known. For example in U.S. Pat. No. 5,151,369 there is disclosed a method of red blood cell lysing and hemoglobin denaturing in which lithium salts, particularly lithium thiocyanate, are used.